Fluids 4 Samples Obtained from Cell Cultures

Samples obtained from such a culture system should be processed by the two-step procedure described above to obtain the individual cells, free from the extracellular compartment. However, note that with cultured samples there is an additional extracellular compartment, which is the culture fluid used to support the growth of the sample. It is not at all uncommon to find enzymatic activities in this fluid. Some of these are normal constituents of the culture medium, while some are a consequence of growth of the sample. 

Any proteomic study starts with the collection of proteins from biological samples such as cell culture media, cultured cells, serum, or any biological fluid, and a variety of animal tissues. The first step is to obtain a protein sample under conditions of least protein degradation. This involves use of various protease inhibitors that stop the protein degradation. The use of protease inhibitors depends on the type of sample and the analytical technique used in the subsequent analysis. The selection of protease inhibitors used is critical since many protease inhibitors and detergents used in the preparation of tissue homogenates can interfere with mass spectrometry (MS)... 

A protein is bound to the affinity adsorbent under conditions sufficiently mild to bind as few contaminating proteins as possible. The sample is usually in equilibration buffer and may be either a crude cell extract, culture supernatant fluid or obtained after a precipitation or ion-exchange step. Adsorption is better from more concentrated samples and higher flow rates can be applied, but the concentration is limited by the viscosity of the sample. It is acceptable to apply dilute samples as these are concentrated on the adsorbent. Overall, the volume of sample is not crucial provided that it is not so large that time becomes a limiting factor, unless the protein is weakly... 

The major Impetus to the development of methods for the prenatal detection of genetic disorders derives. In historical terms, from the roughly simultaneous development of three major techniques (11-14). One was the technique, and the willingness to use It, for obtaining samples of amnlotlc fluid early In gestation. The second was the development of techniques for the culture of human cells in vitro, and the third was the development of better techniques for cytogenetic analysis. As will be described below, with the availability of these three techniques It became possible first to work out methods for the examination of fetal chromosomes, and then, by extension, to devise ways of determining other characteristics of the fetus. 

Flow cytometry [141, 142] is a technique that allows the measurement of multiple parameters on individual cells. Cells are introduced in a fluid stream to the measuring point in the apparatus. Here, the cell stream intersects a beam of light (usually from a laser). Light scattered from the beam and/or cell-associated fluorescence are collected for each cell that is analysed. Unlike the majority of spectroscopic or bulk biochemical methods it thus allows quantification of the heterogeneity of the cell sample being studied. This approach offers tremendous advantages for the study of cells in industrial processes, since it not only enables the visualisation of the distribution of a property within the population, but also can be used to determine the relationship between properties. As an example, flow cytometry has been used to determine the size, DNA content, and number of bud scars of individual cells in batch and continuous cultures of yeast [143,144]. This approach can thus provide information on the effect of the cell cycle on observed differences between cells that cannot be readily obtained by any other technique. 

In group I, we considered the question of obtaining an activity from a tissue or organ, from a biological fluid, or from cultured cells. The primary task in all these samples was the separation of the extracellular and cellular compartments. Next, the problem of separation of the different cell types within the cellular compartment was considered. In the section that follows, we will open the cell for a look inside. However, let us first consider briefly the surface of the intact cell and the problems associated with the assay of any activities that might be located there. 

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